U.S. patent application number 13/582179 was filed with the patent office on 2013-01-03 for drug for inflammatory bowel disease.
This patent application is currently assigned to DAINIPPON SUMITOMO PHARMA CO., LTD.. Invention is credited to Takamasa Watanabe.
Application Number | 20130004516 13/582179 |
Document ID | / |
Family ID | 44542285 |
Filed Date | 2013-01-03 |
United States Patent
Application |
20130004516 |
Kind Code |
A1 |
Watanabe; Takamasa |
January 3, 2013 |
DRUG FOR INFLAMMATORY BOWEL DISEASE
Abstract
Anti-CD81 antibody displays not only a remission induction
effect, but also a long-term remission maintenance effect on
inflammatory bowel disease when administered in a single dose, and
is therefore effective as a drug for maintaining remission from
inflammatory bowel disease. Moreover, anti-CD81 antibody is
effective as a drug for the prevention, amelioration and treatment
of inflammatory bowel disease because it has preventive,
therapeutic and ameliorating effects on refractory inflammatory
bowel disease.
Inventors: |
Watanabe; Takamasa;
(Osaka-shi, JP) |
Assignee: |
DAINIPPON SUMITOMO PHARMA CO.,
LTD.
Osaka-shi, Osaka
JP
|
Family ID: |
44542285 |
Appl. No.: |
13/582179 |
Filed: |
March 3, 2011 |
PCT Filed: |
March 3, 2011 |
PCT NO: |
PCT/JP2011/054900 |
371 Date: |
August 31, 2012 |
Current U.S.
Class: |
424/172.1 |
Current CPC
Class: |
A61K 2039/545 20130101;
A61P 1/04 20180101; C07K 16/2896 20130101; A61P 1/00 20180101; A61K
2039/505 20130101 |
Class at
Publication: |
424/172.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 1/04 20060101 A61P001/04; A61P 1/00 20060101
A61P001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 4, 2010 |
JP |
2010-048052 |
Claims
1. A method for maintaining remission of inflammatory bowel
disease, comprising administering an anti-CD81 antibody to a
subject in need thereof.
2. A method for preventing, ameliorating, or treating refractory
inflammatory bowel disease, comprising administering an anti-CD81
antibody to a subject in need thereof.
3. The method according to claim 2, wherein the refractory
inflammatory bowel disease refers to inflammatory bowel disease
with repeated relapse or recurrence or inflammatory bowel disease
with chronically persistent disease activity.
4. The method according to claim 2, wherein the refractory
inflammatory bowel disease is a steroid-dependent or
steroid-resistant inflammatory disease.
5. The method according to claim 2, wherein the refractory
inflammatory bowel disease refers to inflammatory bowel disease
that is resistant or tolerant to usual treatment.
6. The method according to claim 5, wherein the usual treatment
refers to administration of an existing therapeutic drug.
7. The method according to claim 6, wherein the existing
therapeutic drug is a 5-aminosalicylic acid preparation, steroid,
an immunosuppressant, a TNF inhibitor, or an integrin
inhibitor.
8. The method according to claim 7, wherein the existing
therapeutic drug is sulfasalazine, an anti-TNF antibody,
salazosulfapyridine, mesalazine, betamethasone, betamethasone
sodium phosphate, betamethasone phosphate, prednisolone,
azathioprine, tacrolimus, 6-mercaptopurine, cyclosporine,
infliximab, adalimumab, certolizumab, pegol, or natalizumab.
9. The method according to claim 1, wherein a long-term
remission-maintaining effect is exerted by single-dose
administration.
10. The method according to claim 9, wherein the
remission-maintaining effect is an effect of maintaining a state of
remission by inhibiting repetition of relapse or recurrence of
intestinal inflammation.
11. The method according to claim 1, wherein an anti-CD81 antibody
is administered to a patient with inflammatory bowel disease in
whom remission cannot be maintained by single-dose administration
or even by multiple-dose administration of an existing therapeutic
drug.
12. The method according to claim 11, wherein the existing drug is
5-aminosalicylic acid preparation, steroid, an immunosuppressant, a
TNF inhibitor, an integrin inhibitor, sulfasalazine, an anti-TNF
antibody, salazosulfapyridine, mesalazine, betamethasone,
betamethasone sodium phosphate, betamethasone phosphate,
prednisolone, azathioprine, tacrolimus, 6-mercaptopurine,
cyclosporine, infliximab, adalimumab, certolizumab, pegol, or
natalizumab.
13. The method according to claim 1, wherein the anti-CD81 antibody
is administered once every 2 to 30 weeks during a period of
treatment.
14. The method according to claim 13, wherein a dose per
administration is 0.01 to 15 mg/kg.
15. The method according to claim 1, wherein the inflammatory bowel
disease is ulcerative colitis.
16. The method according to claim 1, wherein the inflammatory bowel
disease is Crohn's disease.
Description
TECHNICAL FIELD
[0001] The present invention relates to a drug for maintaining
remission of inflammatory bowel disease (hereinbelow, may be
abbreviated as "IBD") that contains an anti-CD81 antibody as the
active ingredient or a preventive, ameliorating, or therapeutic
drug for refractory inflammatory bowel disease that contains an
anti-CD81 antibody as the active ingredient. The present invention
further relates to a method for maintaining remission of
inflammatory bowel disease using an anti-CD81 antibody or a method
for preventing, ameliorating, or treating refractory inflammatory
bowel disease using an anti-CD81 antibody.
BACKGROUND ART
[0002] The intestine is the organ where nutrients and water, which
are essential for vital activities of the living body, are digested
and absorbed. Meanwhile, the intestine also has an immune defense
function to get rid of foreign objects such as pathogens, and in
this way it also serves as the organ that governs the maintenance
of life by controlling the contrasting properties in a balanced
manner. However, it is known that once the above functional balance
is troubled, the state of dynamic equilibrium is disturbed, causing
various intestinal diseases. Particularly, recently, an increase
has been observed in the number of patients with inflammatory bowel
disease (abbreviated as IBD), which is a disease that causes
gastrointestinal abnormalities such as abdominal pain, diarrhea,
and bloody stool with repeated relapse and remission. IBD is
classified into ulcerative colitis (abbreviated as UC) and Crohn's
disease (abbreviated as CD) based on its pathological
condition.
[0003] UC is a disease that is mainly characterized by diffuse
inflammation of the intestinal mucosa that is localized in the
large intestine, and repeated inflammation leads to the development
of colorectal cancer, which often ends up with the need for
surgery. Even after surgery, there are problems of increased bowel
movements, fecal incontinence, and development of pouchitis. With
regard to UC, the following types of symptoms are known;
first-attack-only type, relapse-remitting type, chronic-continuous
type, and acute-fulminating type. While steroid is administered to
severe cases of UC, more attentive care needs to be paid to the
development of side effects (osteoporosis and susceptibility to
infection) caused by steroid administration, etc., and thus it is
advisable to keep in mind that unnecessarily prolonged steroid
administration should be avoided.
[0004] CD is a lesion spanning from the small intestine to the
large intestine, and it is a disease involving intense transmural
inflammation mainly under discontinuous mucus, and repeated
inflammation causes intestinal complications (stricture, fistulas,
and abscess), leading to the need for surgery (Non Patent
Literature 1).
[0005] The remission phase means not the curing of disease but the
period in which the symptoms are subsided. For refractory diseases
for which complete healing is hard to achieve like IBD, how long
the period in which the symptoms are subsided can be maintained
becomes important. With the current drug therapy, relapse of IBD is
observed at a ratio of a certain number of patients not only after
discontinuation of administration of drugs, but also during the
administration of drugs, and in many cases it merely aims at the
inhibition of the secretion and the action of inflammatory factors
as a way of symptomatic treatment. Recently, there are cases in
which anti-TNF-.alpha. antibody (Remicade or infliximab) is used
for refractory IBD, in which obvious clinical symptoms that are
attributable to the disease persist even after the implementation
of proper treatment such as nutrition therapy and other drug
therapies (such as a 5-aminosalicylic acid preparation). However,
there is a report that relapse of disease is observed after
single-dose administration of anti-TNF-.alpha. antibody, and even
while the administration is continued, relapse is still observed in
some patients (Non Patent Literature 2 and Non Patent Literature
3).
[0006] Meanwhile, CD81 is a 26 kDa surface molecule that is
expressed in a wide variety of cells, and it acts to reduce the
threshold for B cell activation by forming a complex with CD21,
CD19, and Leu13 on B cells. On T cells, it assembles with CD4 and
CD8 to transmit the stimulus information into the cell. Further, it
is physiologically and functionally associated with various kinds
of integrins to activate VLA-4 (.alpha.4.beta.1 integrin) on B
cells and LFA-1 (.alpha.L.beta.2 integrin) on thymocytes.
[0007] Pertaining to the anti-CD81 antibody, there is a report that
single-dose administration of an anti-CD81 antibody inhibits 45
hours of the vascular permeability response that occurs in the
model of passive cutaneous anaphylaxis, which is an allergic
disease (Patent Literature 1). Also, there is a report that
administration of an anti-CD81 antibody successfully inhibits 5
days of symptoms of single inflammatory lesions in the intestine
that develop in the mouse colitis model, which is an animal model
of inflammatory bowel disease (Patent Literature 2).
[0008] Further, there is also a report that symptoms of
neuroparalysis with a single lesion that develop in the
experimental autoimmune encephalomyelitis mouse, which is a model
animal of multiple sclerosis, were successfully inhibited by a
total of 10 times of every-other-day administration of Eat2, which
is a clone of an anti-CD81 antibody, whereas the symptoms could not
be inhibited by 2F7, which is another clone of an anti-CD81
antibody (Non Patent Literature 4).
[0009] As described above, a disease model in which amelioration is
achieved with every-other-day administration of an anti-CD81
antibody has been reported, indicating that continuous
administration of an anti-CD81 antibody is important for exertion
of the efficacy.
CITATION LIST
Patent Literature
[0010] Patent Literature 1: International Publication No. WO
1998/025647 [0011] Patent Literature 2: International Publication
No. WO 2005/021792
Non Patent Literature
[0011] [0012] Non Patent Literature 1: Inflamm. Bowel. Dis. 8, 244
to 250, 2002 [0013] Non Patent Literature 2: New England Journal of
Medicine. 337, 1029 to 1035, 1997 [0014] Non Patent Literature 3:
Journal of Clinical Gastroenterology. 41; 799 to 809, 2007 [0015]
Non Patent Literature 4: Neurobiology of Disease 31, 413 to 421,
2008
SUMMARY OF INVENTION
Technical Problem
[0016] Creation of a drug capable of preventing, treating, or
ameliorating the symptoms of refractory inflammatory bowel disease
with repeated remission and relapse (recurrence) and a drug capable
of maintaining remission of the symptoms of such disease is
awaited. In other words, the current situation is that no progress
has been made in the development of a drug for maintaining
remission of inflammatory bowel disease for improvement of
long-term prognosis. In light of the above, creation of a drug for
improving long-term prognosis, particularly, a drug capable of
reducing recurrence and surgery is demanded.
[0017] Accordingly, the present invention aims to provide a drug
for maintaining remission of inflammatory bowel disease having not
only a remission-inducing effect, but also a long-term
remission-maintaining effect, and a method for maintaining
remission of inflammatory bowel disease. The present invention
further aims to provide a preventive, ameliorating, or therapeutic
drug for refractory inflammatory bowel disease and a method for
preventing, ameliorating, or treating refractory inflammatory bowel
disease.
Solution to Problem
[0018] The present inventors conducted an intensive research to
achieve the aforementioned goals. As a result, they have found that
an anti-CD81 antibody has not only a remission-inducing effect, but
also a long-term remission-maintaining effect, and thus is useful
as a drug for maintaining remission of inflammatory bowel disease,
and also as a preventive, ameliorating, or therapeutic drug for
refractory inflammatory bowel disease among other types of
inflammatory bowel disease.
[0019] The present invention was completed based on the foregoing
findings.
[0020] That is, the present invention relates to the following [1]
to [32]:
[1]. a drug for maintaining remission of inflammatory bowel
disease, comprising an anti-CD81 antibody as an active ingredient;
[2]. a preventive, ameliorating, or therapeutic drug for refractory
inflammatory bowel disease, comprising an anti-CD81 antibody as an
active ingredient; [3]. the preventive, ameliorating, or
therapeutic drug according to the aforementioned [2], wherein the
refractory inflammatory bowel disease refers to inflammatory bowel
disease with repeated relapse or recurrence or inflammatory bowel
disease with chronically persistent disease activity; [4]. the
preventive, ameliorating, or therapeutic drug according to the
aforementioned [2] or [3], wherein the refractory inflammatory
bowel disease is a steroid-dependent or steroid-resistant
inflammatory disease; [5]. the preventive, ameliorating, or
therapeutic drug according to any one of the aforementioned [2] to
[4], wherein the refractory inflammatory bowel disease refers to
inflammatory bowel disease that is resistant or tolerant to usual
treatment; [6]. the preventive, ameliorating, or therapeutic drug
according to the aforementioned [5], wherein the usual treatment
refers to administration of an existing therapeutic drug; [7]. the
preventive, ameliorating, or therapeutic drug according to the
aforementioned [6], wherein the existing therapeutic drug is a
5-aminosalicylic acid preparation, steroid, an immunosuppressant, a
TNF inhibitor, or an integrin inhibitor; [8]. the preventive,
ameliorating, or therapeutic drug according to the aforementioned
[7], wherein the existing therapeutic drug is sulfasalazine, an
anti-TNF antibody, salazosulfapyridine, mesalazine, betamethasone,
betamethasone sodium phosphate, betamethasone phosphate,
prednisolone, azathioprine, tacrolimus, 6-mercaptopurine,
cyclosporine, infliximab, adalimumab, certolizumab, pegol, or
natalizumab; [9]. the drug according to any one of the
aforementioned [1] to [8], wherein the drug has a long-term
remission-maintaining effect by single-dose administration; [10].
the drug according to the aforementioned [9], wherein the
remission-maintaining effect is an effect of maintaining a state of
remission by inhibiting repetition of relapse or recurrence of
intestinal inflammation; [11]. the drug according to any one of the
aforementioned [1] to [4], wherein the drug is administered to a
patient with inflammatory bowel disease in whom remission cannot be
maintained by single-dose or even by multiple-dose administration
of an existing therapeutic drug; [12]. the drug according to the
aforementioned [11], wherein the existing drug is the existing drug
according to the aforementioned [7] or [8]; [13]. the drug
according to any one of the aforementioned [1] to [12], wherein the
drug is administered once every 2 to 30 weeks during a period of
treatment; [14]. the drug according to the aforementioned [13],
wherein a dose per administration is 0.01 to 15 mg/kg; [15]. the
drug according to any one of the aforementioned [1] to [14],
wherein the inflammatory bowel disease is ulcerative colitis; [16].
the drug according to any one of the aforementioned [1] to [14],
wherein the inflammatory bowel disease is Crohn's disease; [17]. a
method for maintaining remission of inflammatory bowel disease,
comprising administering an anti-CD81 antibody to a subject in need
thereof; [18]. a method for preventing, ameliorating, or treating
refractory inflammatory bowel disease, comprising administering an
anti-CD81 antibody to a subject in need thereof; [19]. the method
according to the aforementioned [18], wherein the refractory
inflammatory bowel disease refers to inflammatory bowel disease
with repeated relapse or recurrence or inflammatory bowel disease
with chronically persistent disease activity; [20]. the method
according to the aforementioned [18] or [19], wherein the
refractory inflammatory bowel disease is a steroid-dependent or
steroid-resistant inflammatory disease; [21]. the method according
to any one of the aforementioned [18] to [20], wherein the
refractory inflammatory bowel disease is inflammatory bowel disease
that is resistant or tolerant to usual treatment; [22]. the method
according to the aforementioned [21], wherein the usual treatment
refers to administration of an existing therapeutic drug; [23]. the
method according to the aforementioned [22], wherein the existing
therapeutic drug is a 5-aminosalicylic acid preparation, steroid,
an immunosuppressant, a TNF inhibitor, or an integrin inhibitor;
[24]. the method according to the aforementioned [23], wherein the
existing therapeutic drug is sulfasalazine, an anti-TNF antibody,
salazosulfapyridine, mesalazine, betamethasone, betamethasone
sodium phosphate, betamethasone phosphate, prednisolone,
azathioprine, tacrolimus, 6-mercaptopurine, cyclosporine,
infliximab, adalimumab, certolizumab, pegol, or natalizumab; [25].
the method according to any one of the aforementioned [17] to [24],
wherein a long-term remission-maintaining effect is exerted by
single-dose administration; [26]. the method according to the
aforementioned [25], wherein the remission-maintaining effect is an
effect of maintaining a state of remission by inhibiting repetition
of relapse or recurrence of intestinal inflammation; [27]. the
method according to any one of the aforementioned [17] to [20],
wherein an anti-CD81 antibody is administered to a patient with
inflammatory bowel disease in whom remission cannot be maintained
by single-dose administration or even by multiple-dose
administration of an existing therapeutic drug; [28]. the method
according to the aforementioned [27], wherein the existing drug is
the existing drug according to the aforementioned [23] or [24];
[29]. the method according to any one of the aforementioned [17] to
[28], wherein the anti-CD81 antibody is administered once every 2
to 30 weeks during a period of treatment; [30]. the method
according to the aforementioned [29], wherein a dose per
administration is 0.01 to 15 mg/kg; [31]. the method according to
any one of the aforementioned [17] to [30], wherein the
inflammatory bowel disease is ulcerative colitis; and [32]. the
method according to any one of the aforementioned [17] to [30],
wherein the inflammatory bowel disease is Crohn's disease.
Advantageous Effects of Invention
[0021] The present invention revealed that an anti-CD81 antibody
was capable of preventing, ameliorating, and treating refractory
inflammatory bowel disease and an anti-CD81 antibody had a
long-term remission-maintaining effect even by single-dose
administration. In other words, the present invention revealed that
an anti-CD81 antibody exerted a profound effect even in a patient
with refractory inflammatory bowel disease (IBD) in whom
single-dose administration or even multiple-dose administration of
an existing therapeutic agent for inflammatory bowel disease has
achieved only as far as a remission-inducing effect.
[0022] Accordingly, a drug containing an anti-CD81 antibody as the
active ingredient exhibits not only a remission-inducing effect on
inflammatory bowel disease, but also a long-term
remission-maintaining effect by single-dose administration, and
thus is utilizable as a drug for maintaining remission and as a
preventive, ameliorating, or therapeutic drug for refractory
inflammatory bowel disease. Further, an anti-CD81 antibody exhibits
not only a remission-inducing effect, but also a long-term
remission-maintaining effect on inflammatory bowel disease by
single-dose administration, and thus single-dose administration of
an anti-CD81 antibody is utilizable as a method for maintaining
remission or a method for preventing, ameliorating, or treating
refractory inflammatory bowel disease.
BRIEF DESCRIPTION OF DRAWINGS
[0023] [FIG. 1] FIG. 1 (a) is a graph showing the chronological
changes in the symptoms of colitis in the mouse model of
relapse-remitting type ulcerative colitis by single-dose
administration of either a hamster IgG antibody or an anti-CD81
antibody in the pharmacological test conducted in Example 1. FIG.
1(b) is a graph showing the chronological changes in the symptoms
of colitis in the mouse model of relapse-remitting type ulcerative
colitis by single-dose administration of either a rat IgG antibody
or a rat anti-TNF-.alpha. antibody or by daily administration of
sulfasalazine in the pharmacological test conducted in Example
1.
[0024] [FIG. 2] FIG. 2 is a graph showing the chronological changes
in the symptoms of colitis in the mouse model of relapse-remitting
type inflammatory bowel disease (Crohn's disease and ulcerative
colitis) by single-dose administration of either a hamster IgG
antibody or an anti-CD81 antibody or by daily administration of
sulfasalazine in the pharmacological test conducted in Example
2.
[0025] [FIG. 3] FIG. 3 is a graph showing the chronological changes
in the symptoms of colitis in the mouse model of relapse-remitting
type ulcerative colitis by single-dose administration of either a
hamster IgG antibody or an anti-CD81 antibody in the
pharmacological test conducted in Example 3.
[0026] [FIG. 4] FIG. 4 is a graph showing the chronological changes
in the symptoms of colitis in the mouse model of relapse-remitting
type ulcerative colitis by single-dose administration of each of
the hamster IgG antibody, the anti-CD81 antibody clone 2F7, and the
anti-CD81 antibody clone Eat2 in the pharmacological test conducted
in Example 4.
DESCRIPTION OF EMBODIMENTS
[0027] The following present invention will be described in
detail.
(1) Anti-CD81 Antibody
[0028] The anti-CD81 antibody in the present invention may be an
antibody that specifically recognizes CD81. Specifically, it may be
an antibody that can specifically recognize the expression product
(protein) of the CD81 gene (which may also be referred to as "CD81"
in the present specification).
[0029] Here, CD81 encompasses not only a "protein" or
"(poly)peptide" expressed by a specific amino acid sequence (SEQ ID
NO: 2 (NP.sub.--004347)) indicating human CD81, but also its
homologues substances (homologues and splice variants), mutants,
derivatives, matured forms, amino acid-modified forms, and the like
as long as they have equivalent biological functions to the
aforementioned protein or (poly)peptide. Here, examples of the
homologue include a protein of other species of organism such as a
mouse or a rat that corresponds to the human protein, which can be
deductively identified based on the base sequence of a gene (SEQ ID
NO: 1 (NM.sub.--004356)) published in http://www.ncbi.nlm.nih.gov.
Also, the mutant encompasses a naturally occurring allelic mutant,
a non-naturally-occurring mutant, and a mutant having a modified
amino acid sequence by artificial deletion, substitution, addition,
or insertion. Also, examples of the aforementioned mutant include
one having at least 70% similarity, preferably 80% similarity, more
preferably 95% similarity, and even more preferably 97% similarity
to the non-mutated protein or (poly)peptide. Also, examples of the
amino acid-modified form encompass a naturally-occurring amino
acid-modified form and a non-naturally-occurring amino
acid-modified form, and specific examples include one having a
phosphorylated amino acid.
[0030] The aforementioned antibody used in the present invention
includes a polyclonal antibody and a monoclonal antibody. Depending
on the type of the constant domain of the heavy chain, an antibody
is classified into five major classes; namely, IgA, IgD, IgE, IgG
or IgM. Some of them are further classified into subclasses or
isotypes such as IgG1, IgG2, IgG3, and IgG4.
[0031] The antibody of the present invention is preferably a
monoclonal antibody, and it encompasses a human antibody, a
chimeric antibody, a humanized antibody, a single chain antibody,
or a Fab fragment and a fragment produced by a Fab expression
library, a low molecular weight antibody (including a fragment of
an antibody), a polyspecific antibody, and further, a modified
antibody. The antibody of the present invention also encompasses a
part of the aforementioned antibody that has an antigen-binding
property.
[0032] The phrase "specifically recognize" refers to binding to
CD81.
[0033] As the anti-CD81 antibody used in the present invention, for
example, a commercially available anti-CD81 antibody (the product
of Pharmingen, Santa Cruz Biotechnology, Inc., BioLegend, Inc.,
SouthernBiotech, Ancell Corporation, MorphoSys AG, Chemicon, Abeam
plc., ImmunoTech. Co., Ltd., R&D, and so on) may be used, while
a polyclonal antibody or a monoclonal antibody produced by publicly
known means may also be used.
[0034] As the anti-CD81 antibody used in the present invention,
particularly, mammal-derived monoclonal and polyclonal antibodies
are preferred. The monoclonal and polyclonal antibodies can be
produced by a method publicly known to those skilled in the
art.
[0035] Examples of the mammal-derived monoclonal and polyclonal
antibodies include one produced in the blood of an animal, one
produced by a hybridoma, one produced by a host transformed with an
expression vector carrying the gene of the antibody by genetic
engineering techniques, one obtained by mass-producing, in the CHO
cell factory, the gene of the most suitable antibody that is
screened from an enormous clone library consisting of a trillion
molecules using phage display, or a human antibody directly
obtained from a transgenic mouse producing a human antibody.
[0036] The protein that is used as a sensitizing antigen for
acquisition of the anti-CD81 antibody of the present invention for
the production of a polyclonal antibody is not limited by the
animal species from which it is derived, which can be a human, a
mouse, a rat, and the like. However, the protein is preferably
selected in consideration of compatibility with the parent cell to
be used for cell fusion, and it is generally preferably a
mammal-derived protein, and particularly preferably a human-derived
protein. For example, when CD81 is human CD81, a human CD81
protein, a cell expressing human CD81, a partial peptide of human
CD81 can be used. Also, the protein may be a complete protein or a
partial peptide of the protein. Examples of the partial peptide of
the protein include an amino group (N) terminal fragment and a
carboxy (C) terminal fragment of the protein. The anti-CD81
antibody in the present invention refers to an antibody that binds
to the full-length protein or a fragment of the protein.
[0037] For example, a polyclonal antibody can be obtained as
follows. That is, a small animal such as a rabbit is immunized with
a naturally-occurring CD81 protein or a recombinant CD81 protein
that is expressed in a microorganism such as E. coli as a fusion
protein with GST, or a partial peptide thereof, and the serum of
this animal is obtained. The serum thus obtained is purified by,
for example, ammonium sulfate precipitation, a protein A column, a
protein G column, DEAE ion-exchange chromatography, and an affinity
column coupled with a CD81 protein or a synthetic peptide, whereby
a polyclonal antibody is prepared.
[0038] The antigen can be prepared in accordance with, for example,
a method using a baculovirus (for example, International
Publication No. WO 98/46777). When the antigen has low
antigenicity, it may be conjugated to an antigenic macromolecule
such as albumin and then used for immunization.
[0039] As the method for producing a monoclonal antibody, an animal
is immunized with a sensitizing antigen in accordance with a
publicly known method. As a general method, a sensitizing antigen
is administered to a mammal by an intraperitoneal or subcutaneous
injection. Specifically, a sensitizing antigen is diluted to an
appropriate dose and suspended in Phosphate-Buffered Saline (PBS),
physiological saline, and the like, to which an appropriate amount
of a common adjuvant, for example, the Freund's complete adjuvant,
is added as desired, followed by emulsification. The resulting
emulsion is administered to a mammal several times every four to 21
days. Also, when immunizing an animal with a sensitizing antigen,
an appropriate carrier may be used.
[0040] Once a mammal is immunized as described above and an
elevated serum level of the desired antibody is confirmed, immune
cells are collected from the mammal and subjected to cell fusion.
Examples of the particularly preferred immune cell include a spleen
cell. As the counterpart parent cell to be fused with the
aforementioned immune cell, a mammalian myeloma cell is used. As
the myeloma cell, various kinds of publicly known cell lines, for
example, P3U1 (P3-X63Ag8U1), P3 (P3x63Ag8.653) (J. Immunol. (1979)
123, 1548 to 1550), P3x63Ag8U.1 (Current Topics in Microbiology and
Immunology (1978) 81, 1 to 7), NS-1 (Kohler. G and Milstein, C.,
Eur. J. Immunol. (1976) 6, 511 to 519), MPC-11 (Margulies. D. H. et
al., Cell (1976) 8, 405 to 415), SP2/0 (Shulman, M. et al., Nature
(1978) 276, 269 to 270), FO (de St. Groth, S. F. et al., J.
Immunol. Methods (1980) 35, 1 to 21), 5194 (Trowbridge, I. S., J.
Exp. Med. (1978) 148, 313 to 323), and 8210 (Galfre, G et al.,
Nature (1979) 277, 131 to 133) are preferably used.
[0041] The cell fusion of the aforementioned immune cell with the
myeloma cell can basically be performed in accordance with a
publicly known method, for example, the method of Kohler and
Milstein, et al. (Kohler. G and Milstein, C., Methods Enzymol.
(1981) 73, 3 to 46).
[0042] More specifically, the aforementioned cell fusion is
performed, for example, in a common nutrient liquid culture medium
in the presence of a cell fusion promoter. As the fusion promoter,
for example, polyethylene glycol (PEG) and a hemagglutinating virus
of Japan (HVJ) are used, and further, an auxiliary agent such as
dimethyl sulfoxide may be added to increase the fusion efficiency
as desired.
[0043] The ratio between the immune cell and the myeloma cell used
can be arbitrarily set. For example, it is preferable to use one to
10 times the number of the immune cell than the myeloma cell. As
the liquid culture medium used for the aforementioned cell fusion,
for example, an RPMI 1640 medium, which is suitable for
proliferation of the aforementioned myeloma cell line, a MEM
medium, and in addition, common liquid culture media used for
culturing these kinds of cells can be used, and further, a serum
supplement such as fetal calf serum (FCS) can be used together.
[0044] In cell fusion, certain amounts of the aforementioned immune
cells and myeloma cells are mixed well in the aforementioned liquid
culture medium, to which a PEG solution (for example, an average
molecular weight of approximately 1000 to 6000) that has been
heated to approximately 37.degree. C. in advance, is added at a
concentration of, normally, 30 to 60% (w/v), followed by mixing to
form the desired hybridomas. Subsequently, an appropriate liquid
culture medium is sequentially added, followed by centrifugation to
remove a supernatant. This operation is repeated to remove a cell
fusion agent and the like that are unfavorable for the growth of
the hybridomas.
[0045] The hybridomas obtained as above are screened by culturing
in a common selection medium, for example, a HAT medium (a liquid
culture medium containing hypoxanthine, aminopterin, and
thymidine). This culturing in a HAT medium is continued long enough
(normally, several days to several weeks) for cells other than the
desired hybridomas (unfused cells) to die out. Subsequently, common
limiting dilution technique is applied to screen for a hybridoma
producing the desired antibody, from which a single clone is
produced.
[0046] In addition to obtaining the aforementioned hybridomas by
immunizing an animal other than the human with an antigen, it is
also possible to sensitize human lymphocytes, for example, EB
virus-infected human lymphocytes, in vitro with a protein, a
protein-expressing cell, or a lysed product of such a cell, and the
sensitized lymphocytes are fused with immortalized human-derived
myeloma cells, for example U266, to obtain a hybridoma producing a
human antibody having the desired activity (for example, a cell
migration-inhibiting activity).
[0047] The monoclonal antibody-producing hybridomas thus
constructed can be subcultured in a common liquid culture medium,
or can be stored for a prolonged period of time in liquid nitrogen.
That is, the monoclonal antibody-producing hybridomas can be
produced by, using the desired antigen or a cell expressing the
desired antigen as the sensitizing antigen, immunizing a subject
with this sensitizing antigen in accordance with a common
immunization method; fusing the immune cell thus obtained with a
publicly known parent cell by a common cell fusion method; and
screening for a monoclonal antibody-producing cell (hybridoma) by a
common screening method. In order to obtain the monoclonal antibody
from the hybridoma, a method of culturing the hybridoma by a common
method and obtaining the antibody as a culture supernatant, or a
method of administering the hybridoma to a mammal compatible with
the hybridoma, allowing the hybridoma to proliferate, and obtaining
the antibody as ascites is adopted. The former method is suitable
for obtaining high-purity antibodies, whereas the latter method is
suitable for a large scale production of antibodies.
[0048] The human antibody refers to an antibody that is the
expression product of the gene of the human-derived antibody. The
human antibody can be obtained by, for example, administering an
antigen to a transgenic animal made capable of producing a
human-derived antibody through introduction of the human antibody
gene locus. Examples of the transgenic animal include a mouse, and
a method for producing a mouse capable of producing the human
antibody is described in, for example, International Publication
No. WO02/43478.
[0049] The monoclonal antibody according to the present invention
also encompasses a monoclonal antibody composed of a heavy chain
and/or light chain having an amino acid sequence resulting from
deletion, substitution, or addition of one or several amino acids
introduced in each of the amino acid sequences of the heavy chain
and/or light chain composing the antibody. These partial
modifications of amino acid (deletion, substitution, insertion, and
addition) can be introduced in the amino acid sequence of the
antibody of the present invention by partially modifying the base
sequence encoding the amino acid sequence. Such partial
modification of base sequence can be introduced by a routine method
employing known site specific mutagenesis (Proc. Natl. Acsd. Sci.
USA., 1984 Vol 81, 5662; Sambrook et al., Molecular Cloning A
Laboratory Manual (1989) Second edition, Cold Spring Harbor
Laboratory Press).
[0050] In the present invention, a gene recombinant antibody that
has been artificially modified for the purpose of lowering
heterologous antigenicity against humans, etc. such as a chimeric
antibody and a humanized antibody can also be used. These modified
antibodies can be produced using known methods.
[0051] A chimeric antibody is an immunoglobulin molecule
characterized by binding of two or more moieties derived from
heterologous animal species. Generally, the variable region (V
region) of a chimeric antibody is derived from a mammalian antibody
other than the human (such as a mouse monoclonal antibody), and the
immunoglobulin constant region (C region) of a chimeric antibody is
derived from a human immunoglobulin molecule. Preferably, a
variable region having low antigenicity is selected, which is
combined with a human constant region similarly having low
antigenicity. Preferably, the resulting combination similarly has
low antigenicity. The chimeric antibody encompasses monovalent
immunoglobulin, bivalent immunoglobulin, and polyvalent
immunoglobulin. The monovalent chimeric antibody is a dimer (HL)
formed by a chimeric H chain bound to a chimeric L chain via a
disulfide bridge. The bivalent antibody is a tetramer (H2L2) formed
by two HL dimers bound together via at least one disulfide
bridge.
[0052] The chimeric antibodies and the methods for producing the
same are already described in the art (Morrison et al., Proc. Natl.
Acad. Sci. USA 81: 6851 to 6855 (1984); Boulianne et al., Nature
312: 643 to 646 (1984); Liu et al., Proc. Natl. Acad. Sci. USA 84:
3439 to 3443 (1987); Sun et al., Proc. Natl. Acad. Sci. USA, 84:
214 to 218 (1987); Better et al., Science 240: 1041 to 1043 (1988);
and Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL Cold Spring
Harbor Laboratory(1988)).
[0053] The humanized antibody, which is also called a reshaped
human antibody, is made by grafting the complementarity determining
region (CDR) of an antibody of a mammal other than the human, for
example a mouse antibody, into the complementarity determining
region of a human antibody. General gene recombination technology
for this is also known (see European Patent Application Publication
No. EP 125023 and International Publication No. WO92/19759). As the
method for producing a humanized antibody, a publicly known method
can be used. For example, a DNA sequence designed to link the CDR
of a mouse antibody and the framework region (FR) of a human
antibody is synthesized by the PCR method from several
oligonucleotides produced in such a way as to have sections
overlapping with one another at the ends thereof. The DNA thus
obtained is ligated to the DNA encoding the C region of a human
antibody, and the resulting ligated DNA is inserted into an
expression vector, which is then introduced into a host so that it
will produce the antibody, whereby the humanized antibody is
obtained (see European Patent Application Publication No. EP 239400
and International Publication No. WO92/19759). The framework region
of a human antibody to be linked via CDR is selected so that the
complementarity determining region forms a favorable
antigen-binding site. If necessary, amino acids in the framework
region of the variable region in the antibody may be substituted so
that the complementarity determining region of a reshaped human
antibody forms an appropriate antigen-biding site (Sato, K. et al.,
Cancer Res. (1993) 53, 851 to 856).
[0054] For a chimeric antibody and a humanized antibody, the human
antibody C region is used. Examples of the human antibody C region
include C.gamma., and for example, C.gamma.1, C.gamma.2, C.gamma.3,
or C.gamma.4 can be used. Also, in order to improve the stability
of the antibody or the production of the antibody, the human
antibody C region may be modified. A chimeric antibody is composed
of the variable region of an antibody derived from a mammal other
than the human and the C region derived from a human antibody, and
a humanized antibody is composed of the complementarity determining
region of an antibody derived from a mammal other than the human
and the framework region and C region derived from a human
antibody. Because these antibodies have reduced antigenicity in the
human body, they are useful as the antibody to be used in the
present invention.
[0055] The antibody used in the present invention may be a fragment
of an antibody or a modified antibody as long as it is preferably
used in the present invention. Examples of the fragment of an
antibody include Fab, F(ab')2, Fv, or a single chain Fv (scFv)
diabody in which Fv of the H and L chains is linked via an
appropriate linker, a low molecular weight antibody, and a single
chain antibody.
[0056] The low molecular weight antibody encompasses a fragment of
an antibody, which is a partially-deleted whole antibody (for
example, whole IgG), and no particularly limitation is imposed as
long as it retains binding ability to the antigen (CD81 protein).
The fragment of an antibody is not particularly limited as long as
it is a part of the whole antibody, and it preferably contains the
heavy chain variable region (VH) or/and the light chain variable
region (VL).
[0057] The single chain antibody is also called "single chain Fv",
namely a "sFv" antibody fragment. It has the VH and VL domains of
an antibody, and these domains are present in a polypeptide single
chain. The "Fv" fragment is the smallest fragment of an antibody,
which contains complete antigen recognition and binding sites.
Generally, an "Fv" fragment is a dimer (VH-VL dimer) in which a VH
and a VL are strongly linked by non-covalent binding. The three
complementarity determining regions (CDRs) of each of the variable
regions interact with each other to form an antigen-binding site on
the surface of the VH-VL dimer. Six CDRs confer the antigen binding
site to an antibody. However, one variable region (or half of the
Fv containing only three antigen-specific CDRs) alone is capable of
recognizing and binding to an antigen, although its affinity is
lower than the affinity of the entire binding site. An Fv
polypeptide further contains a polypeptide linker between the VH
and VL domains so that sFv forms the desired configuration for
antigen binding. (Pluckthun, The Pharmacology of Monoclonal
Antibodies, vol. 113, Rosenberg and Moore eds. Springer-Verlag, New
York, pp. 269 to 315 (1994)).
[0058] As to the method for producing a low molecular weight
antibody and a single chain antibody, for example, a fragment of an
antibody is produced by treating an antibody with an enzyme such as
papain and pepsin, or by constructing a gene encoding such a
fragment of an antibody, introducing the resulting gene into an
expression vector, and then expressing the gene in an appropriate
host cell (for example, see Co, M. S. et al., J. Immunol. (1994)
152, 2968 to 2976, Better, M. & Horwitz, A. H. Methods in
Enzymology (1989) 178, 476 to 496, Plueckthun, A. & Skerra, A.
Methods in Enzymology (1989) 178, 497 to 515, Lamoyi, E., Methods
in Enzymology (1986) 121, 652 to 663, Rousseaux, J. et al., Methods
in Enzymology (1986) 121, 663 to 669, and Bird, R. E. et al.,
TIBTECH (1991) 9, 132 to 137).
[0059] ScFv is obtainable by linking the H chain V region and the L
chain V region of an antibody. These regions are present in a
single polypeptide chain. Generally, an Fv polypeptide further
contains a polypeptide linker between VH and VL, whereby scFv can
form a structure necessary for antigen binding (for general reviews
of scFv, see Pluckthun "The Pharmacology of Monoclonal Antibodies"
Vol. 113 (Rosenberg and Moore eds. (Springer Verlag, New York) pp.
269 to 315, 1994)). The linker is not particularly limited as long
as it does not inhibit the expression of the antibody variable
regions linked to both ends thereof.
[0060] In such scFv, the H chain V region and the L chain V region
are linked via a linker, preferably by a peptide linker (Huston, J.
S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879 to 5883).
The H chain V region and the L chain V region in scFv may be
derived from any of the substances listed as an antibody above. As
the peptide linker linking the V regions, for example, any single
chain peptide consisting of 12 to 19 amino acid residues is
used.
[0061] DNA encoding scFv is obtained as follows; using DNA encoding
the H chain or the H chain V region of an antibody and DNA encoding
the L chain or the L chain V region of an antibody as described
above as a template, the segment of DNA encoding the desired amino
acid sequences within the sequences of the above parts of an
antibody is amplified by PCR using a pair of primers that define
both ends of the DNA. Subsequently, amplification is performed by
combining DNA encoding the peptide linker moiety and a pair of
primers that define so that each of the both ends of the DNA is
linked to the H chain or the L chain, respectively. Further, once
the DNA encoding scFv is produced, an expression vector carrying
the DNA and a host transformed with the expression vector can be
obtained in accordance with a routine method, and scFv can be
obtained in accordance with a routine method by using the host thus
obtained.
[0062] A diabody refers to a bivalent antibody fragment constructed
by gene fusion (for example, Holliger, P. et al., Proc. Natl. Acad.
Sci. USA 90: 6444 to 6448 (1993), EP Patent Application Publication
No. 404097, and International Publication No. WO93/11161). A
diabody is a dimer composed of two polypeptide chains, and normally
in each polypeptide chain, VL and VH are linked by a linker that is
short enough to prevent them from binding to each other within the
same chain, for example, a linker of approximately five residues.
The VL and VH encoded on the same polypeptide chain form a dimer
since the linker between them is too short to allow them to form a
single chain variable region fragment. Consequently, a diabody
possesses two antigen-binding sites.
[0063] Also, sc(Fv)2 is a low molecular weight antibody in which
two VHs and two VLs are linked together by a linker and the like
into a single chain (Hudson et al., J. Immunol. Methods 1999; 231:
177 to 189). The sc(Fv)2 can be produced by, for example, linking
scFvs by a linker.
[0064] In the present invention, as the linker linking the variable
regions of an antibody, any peptide linker that can be introduced
by genetic engineering or a synthetic compound linker, i.e., a
linker disclosed in (for example, see Protein Engineering, 9 (3),
299 to 305, 1996), and the like can be used. When a peptide linker
is used, the length of the linker is not particularly limited, and
it can be appropriately selected by those skilled in the art
according to the purpose; however, normally, it consists of one to
100 amino acids, preferably three to 50 amino acids, more
preferably five to 30 amino acids, and particularly preferably 12
to 18 amino acids (for example, 15 amino acids). The synthetic
compound linker (chemical cross-linking agent) is a cross-linking
agent normally used for cross-linking of peptides, and examples
thereof include N-hydroxysuccinimide (NHS), disuccinimidyl suberate
(DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl
propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP),
ethylene glycol bis(succinimidyl succinate) (EGS) ethylene glycol
bis(sulfosuccinimidyl succinate) (sulfa-EGS), disuccinimidyl
tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),
bis[2-(succinimidooxycarbonyloxy)ethyl] sulphone (BSOCOES), and
bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulphone
(sulfo-BSOCOES), and these cross-linking agents are commercially
available.
[0065] These fragments of an antibody can be produced by acquiring
the gene thereof and expressing it in a host in a similar manner to
the above. The "antibody" as referred to in the claims of the
present application encompasses these fragments of an antibody.
[0066] As a modified antibody, an antibody conjugated with various
kinds of molecules such as polyethylene glycol (PEG) can also be
used. The "antibody" as referred to in the claims of the present
application also encompasses these modified antibodies. Theses
modified antibodies can be obtained by subjecting the antibody thus
obtained to chemical modification. The method of antibody
modification has already been established in the art.
[0067] The antibody produced and expressed as described above can
be isolated from the inside and outside of the cell as well as from
the host, and purified to homogeneity. The antibody used in the
present invention can be isolated and purified by affinity
chromatography. Examples of the column used for affinity
chromatography include a protein A column and a protein G column.
Examples of the carrier used in the protein A column include Hyper
D, POROS, and Sepharose F.F. Besides the above, common methods of
isolation and purification of protein may be used, and no
limitation is imposed on the method.
[0068] For example, the antibody used in the present invention can
be isolated and purified by appropriately selecting and combining
chromatography other than the aforementioned affinity
chromatography, a filter, ultrafiltration, salting-out, dialysis,
and the like. Examples of the chromatography include ion exchange
chromatography, hydrophobic chromatography, and gel filtration. The
above chromatography can be applied to High performance liquid
chromatography (HPLC). Further, reverse-phase HPLC may also be
used.
(2) Inflammatory Bowel Disease
[0069] Inflammatory bowel disease is a disease that causes
intestinal inflammation with repeated severe abdominal pain and
diarrhea. Inflammatory bowel disease is classified into Ulcerative
Colitis (abbreviated as UC) and Crohn's disease (abbreviated as CD)
according to its pathological condition, and in both cases the
disease persists for a long time and often involves repeated
remission and relapse.
[0070] At present, regarding the indication of a therapeutic
pharmaceutical product for inflammatory bowel disease, for example,
azathioprine, which is an immunosuppressant, is approved to be used
for "remission induction and remission maintenance of
steroid-dependent Crohn's disease and remission maintenance of
steroid-dependent ulcerative colitis" in Japan, and tacrolimus,
which is an immunosuppressant, is approved to be used for
"refractory (steroid-resistant and steroid-dependent) active
ulcerative colitis (limited to moderate to severe UC)" in Japan.
Also, for Crohn's disease, it is specified that infliximab, which
is an anti-TNF antibody, "should be administered when obvious
clinical symptoms that are attributable to the disease persist even
after the implementation of proper treatment such as nutrition
therapy and other drug therapies (such as a 5-aminosalicylic acid
preparation)" in Japan. Further, in the U.S., infliximab is
approved to be used for Crohn's disease "for inhibition, remission
induction, and remission maintenance of signs and symptoms in
adults with moderate to severe Crohn's disease that is resistant to
usual treatment and in patients with juvenile active Crohn's
disease, and for reduction in the number of enterocutaneous fistula
and rectovaginal fistula and for maintenance of fistula closure in
patients with adult Crohn's disease with fistula", and for
ulcerative colitis, "for inhibition of signs and symptoms as well
as induction and maintenance of clinical remission and mucosal
healing in patients with moderate to severe active ulcerative
colitis that is resistant to usual treatment."
[0071] As described above, in the treatment of inflammatory bowel
disease, the usage of a therapeutic pharmaceutical product is
approved for remission induction and remission maintenance, and the
states of disease are distinguished in terms of the concept of
severity of disease (mild to severe), the presence or absence of
resistance to existing drugs, intractability, and the like. Here,
remission induction refers to inducing remission in the active
lesions involving bloody stool, ulcer and erosion in the intestinal
tract, reduced QOL, and the like. Also, remission maintenance
refers to inhibiting the repetition of relapse/recurrence of the
active lesions and maintaining the remission state for as long as
possible. Here, the term "remission" refers to an improvement in an
activity index, where the activity index is obtained by expressing
the disease activity such as diarrhea, melena, abdominal pain,
pyrexia, body weight reduction, and anal pain, all of which are the
symptoms of inflammatory bowel disease, in terms of Crohn's disease
activity index (CDAI), IOIBD assessment score, Dutch AI, ulcerative
colitis activity index, and the like.
[0072] The term "refractory (intractability)" refers to a state in
which repeated relapses occur and clinical symptoms attributable to
the disease persist, i.e., the disease activity chronically
persists, even with proper implementation of usual treatment such
as nutrition therapy and other drug therapies. The phrase "the
disease activity chronically persists" refers to a state in which
the symptoms persists for six months or more after the initiation
of treatment, and this encompasses a condition (disease) that is
resistant or tolerant to usual treatment such as a
steroid-dependent or steroid-resistant condition or disease.
[0073] Examples of the existing drug used for usual treatment
include a 5-aminosalicylic acid preparation (for example,
salazosulfapyridine, sulfasalazine, and mesalazine), steroid (for
example, betamethasone, betamethasone sodium phosphate,
betamethasone phosphate, and prednisolone), an immunosuppressant
(for example, azathioprine, tacrolimus, 6-mercaptopurine, and
cyclosporine), a TNF inhibitor (for example, infliximab,
adalimumab, certolizumab, pegol), and an integrin inhibitor (for
example, natalizumab).
[0074] The present invention provides a drug containing an
anti-CD81 antibody as the active ingredient that has a long-term
remission-maintaining effect and is effective for ameliorating or
treating relapse-remitting type inflammatory bowel disease (IBD)
among other types of IBD by single-dose administration. In the
present invention, the term "relapse" refers to redevelopment of
symptoms after a certain period of time after the subsidence of the
symptoms of inflammatory bowel disease, and the term "recurrence"
also has the same meaning.
[0075] Patent Literature 2 (International Publication No.
WO/2005/021792) has introduced a novel finding that CD81 is highly
expressed in IBD-pathogenic cells, and an anti-CD81 antibody exerts
a therapeutic effect in the mouse model of first-attack-only type
colitis. However, International Publication No. WO/2005/021792 does
not reveal that an anti-CD81 antibody is also effective for
prevention, amelioration, and treatment of relapse-remitting type
and chronic continuous type refractory inflammatory bowel disease
with repeated relapse (recurrence) and remission. The present
inventors produced a novel animal model presenting with
inflammatory bowel disease accompanying relapse-remitting type
symptoms and newly found that only single-dose administration of an
anti-CD81 antibody to this animal model successfully inhibited
persistence and relapse (recurrence) of intestinal inflammation.
The present invention is based on a novel finding that an anti-CD81
antibody is also effective for prevention, amelioration, and
treatment of relapse-remitting type (symptoms of repeated relapse
(recurrence) and remission) and chronic continuous type refractory
inflammatory bowel disease.
[0076] Accordingly, the drug for maintaining remission of IBD or
the preventive, ameliorating, or therapeutic drug for refractory
IBD provided by the present invention contains an anti-CD81
antibody, which has the remission-maintaining effect that the
existing therapeutic agents do not have. In other words, in a
patient in whom symptoms of repeated relapse (recurrence) and
remission cannot be prevented, ameliorated, or treated by
single-dose or even by multiple-dose administration of existing
therapeutic agents, such symptoms can be prevented, ameliorated, or
treated even by single-dose administration.
[0077] The drug containing an anti-CD81 antibody as the active
ingredient can be administered either orally or parenterally;
however, it is preferably administered parenterally. Specific
examples of the dosage form include an injection form, a nasal
administration form, a pulmonary administration form, and a
transdermal administration form. As an example of the injection
form, the drug can be administered systemically or locally by, for
example, intravenous injection, intramuscular injection,
intraperitoneal injection, and subcutaneous injection. The dosage
varies depending on the kind of active ingredient, the
administration route, the age, body weight, and symptoms of the
subject of administration or the patient, and the like, and
therefore cannot be flatly defined; however, normally, as a dose
per administration, it is administered at 0.01 to 15 mg/kg,
preferably at 1 to 15 mg/kg, more preferably at 3 to 5 mg/kg, or
preferably at 0.01 to 5 mg/kg, more preferably at 0.05 to 5 mg/kg.
As to the administration interval, no subsequent administration may
be given as long as the effect continues. For example, during the
treatment period, it is administered once every 2 to 30 weeks, 2 to
15 weeks, 2 to 13 weeks, 2 to 10 weeks, or 2 to 8 weeks, or it may
be administered at a frequency of once every several weeks to
several months or once every year to several years. In other words,
as a dose per administration, an anti-CD81 antibody is normally
administered at 0.01 to 15 mg/kg, preferably at 1 to 15 mg/kg, more
preferably at 3 to 5 mg/kg, or preferably at 0.01 to 5 mg/kg, more
preferably at 0.05 to 5 mg/kg, thereby exerting a
remission-maintaining effect for as long as 2 to 13 weeks, 2 to 10
weeks, or 2 to 8 weeks. Also, when sufficient efficacy cannot be
obtained or the efficacy is diminished, setting the above dosage at
one cycle, multiple cycles can be administered (for example, 2 to 5
times), whereby the efficacy is achieved and then maintained.
[0078] The drug containing an anti-CD81 antibody as the active
ingredient per se is administered as a drug prepared by a publicly
known drug formulation method. For example, it can be used in the
form of an injection of a sterile solution or a suspension prepared
with water or a pharmaceutically acceptable liquid other than
water. Also, for example, the drug can be prepared by combining the
antibody with a pharmaceutically acceptable carrier or medium,
specifically, sterilized water, physiological saline, an
emulsifier, a suspending agent, a surfactant, a stabilizer, a
vehicle, a preservative, and the like and mixing them in a unit
dosage form required for generally approved practice of drug
manufacture. The amount of the active ingredient in these
preparations is adjusted such that an appropriate amount in the
prescribed range is obtained.
[0079] A sterile composition for injection can be formulated in
accordance with common practice of drug manufacture using a vehicle
such as distilled water for injection. Examples of an aqueous
solution for injection include physiological saline and an isotonic
solution containing glucose and other adjunct agents such as
D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and an
appropriate solubilizing agent such as alcohol, specifically,
ethanol, polyalcohol such as propylene glycol and polyethylene
glycol, and a nonionic surfactant such as polysorbate 80TM and
HCO-50 can be used together.
[0080] Examples of an oily solution include sesame oil and soybean
oil, and these oils may be used with benzyl benzoate and benzyl
alcohol as a solubilizing agent. Also, a buffer such as a phosphate
buffer and a sodium acetate buffer, a soothing agent such as
procaine hydrochloride, a stabilizer such as benzyl alcohol,
phenol, and an antioxidant may be added. The injection solution
thus prepared is normally placed in an appropriate ampoule.
EXAMPLES
[0081] Next, the present invention will be explained further
specifically with reference to Examples. However, the present
invention is not limited in any way by these Examples.
Example 1
Relapse-Inhibitor Effect of Single-Dose Administration of an
Anti-CD81 Antibody in the Mouse Model of Colitis Induced by an
Aqueous Solution of Dextran Sulfate (DSS)
[0082] An aqueous solution of dextran sulfate (DSS) was
administered to a mouse to induce relapse-remitting type ulcerative
colitis. To this mouse model of relapse-remitting type ulcerative
colitis, an anti-CD81 antibody was administered once and a relapse
(recurrence)-inhibitory effect of single-dose administration of an
anti-CD81 antibody on ulcerative colitis was examined by comparing
with single-dose administration of an anti-TNF-.alpha. antibody and
multiple-dose administration of sulfasalazine, both of which are
the existing therapeutic agents.
1. Method
(1) Preparation of DSS
[0083] Dextran sulfate (the product of TdB consultancy AB, an
average molecular weight of 47,000) was dissolved in drinking water
to prepare a 1% aqueous solution.
(2) Preparation of an Administration Solution
[0084] As the test drug to be studied, a hamster anti-CD81
antibody, a rat anti-TNF-.alpha. antibody, and sulfasalazine were
used, and as the pathological condition control test drug, rat IgG
and hamster IgG were used.
[0085] Phosphate buffer was added to the concentrated original
solutions of the hamster anti-CD81 antibody (Clone: 2F7, the
product of SouthernBiotech, a monoclonal antibody for mouse CD81)
and rat IgG and to the original solutions of the rat
anti-TNF-.alpha. antibody and hamster IgG whereby 0.25 mg/ml
administration solutions were prepared. As to sulfasalazine (the
product of Sigma), it was suspended in a 0.5% aqueous solution of
methyl cellulose (the product of Nacalai Tesque, Inc.) in distilled
water for injection (the product of Otsuka Pharmaceutical Co.,
Ltd.) at 20 mg/ml, whereby an administration solution was
prepared.
(3) Grouping, DSS Drug Administration
[0086] Four-week-old mice (BALB/cAnNCrj, male, the product of
Charles River Laboratories Japan, Inc.) were purchased, and the
mice in which no abnormality was found after six days of quarantine
rearing were measured for body weight and then divided into six
groups, where one group contained 10 mice. For five days after
grouping, five groups of mice were fed 1% DSS ad libitum (first DSS
ingestion), while one group of mice were fed normal drinking water
as a normal mouse group. After five days of DSS ingestion, all six
groups of mice were fed normal drinking water for five days. After
ingestion of normal drinking water, five groups of mice were fed 1%
DSS ad libitum (second DSS ingestion), while one group of mice were
fed normal drinking water as a normal mouse group, and the test was
completed. On the day of grouping, hamster IgG rat IgG the
anti-CD81 antibody, and the anti-TNF antibody were
intraperitoneally administered at 0.5 mg/mouse once. From the day
of grouping to the day before completion of the test, the
sulfasalazine administration solution was orally administered once
a day at 200 mg/kg.
(4) Colitis Symptom Score Assessment
[0087] From the day of grouping to the day of completion of the
test, the symptoms of colitis were observed. The symptoms of
colitis were assessed by score according to the condition of stool
(normal stool (score: 0), loose stool (score: 1), and diarrhea
(score: 2)).
(5) Ulcer Score Assessment
[0088] On the day of completion of the experiment, 0.1 ml of a 2.5%
brilliant blue 6B solution was administered to all groups of mice
via tail vein. Ten minutes after the intravenous injection, the
mice were euthanized by carbon dioxide gas, followed by blood
removal by heart puncture. Then, a segment of the large intestine
from 2 cm to 4 cm above the anus was taken out and thoroughly
washed with physiological saline, which was then sandwiched between
filter paper, fixed for 30 minutes in 75% ethanol, and immersed in
physiological saline containing 5% hydrogen peroxide for 10
minutes. Subsequently, the large intestine was thoroughly washed
with an ethanol solution and the size of ulcer was measured. The
mouse with ulcer of 1 mm.sup.2 or larger was assessed as ulcer
score "1", whereas the mouse without ulcer of 1 mm.sup.2 or larger
was assessed as ulcer score "0."
2. Results
(1) Chronological Changes in the Symptoms of Colitis by Colitis
Symptom Score Assessment
[0089] The chronological changes in the symptoms of colitis by
colitis symptom score assessment in the mice that received
single-dose administration of either one of hamster IgG and the
hamster anti-CD81 antibody and in the mice that ingested normal
drinking water (normal group) were shown in FIG. 1(a). Also, the
chronological changes in the symptoms of colitis by colitis symptom
score assessment in the mice that received single-dose
administration of either one of rat IgG and the rat
anti-TNF-.alpha. antibody and in the mice that received daily
administration of sulfasalazine were shown in FIG. 1(b). In FIG.
1(a) and FIG. 1(b), Day 0 corresponds to the day of grouping, Days
0 to 4 correspond to the first DSS ingestion period, Days 5 to 9
correspond to the normal drinking water ingestion period, and Days
10 to 14 correspond to the second DSS ingestion period.
[0090] As is apparent from the hamster IgG administration group in
FIG. 1(a) and the rat IgG administration group in FIG. 1(b), the
first peak of the symptoms of colitis (first episode of colitis)
after the first DSS ingestion was observed from Day 5 to Day 9 in
FIG. 1(a) and FIG. 1(b), and the second symptoms of colitis at
relapse (relapse colitis) were observed from Day 10 to Day 14 in
FIG. 1(a) and FIG. 1(b). Also, compared to the inhibitory effect of
daily administration of sulfasalazine and single-dose
administration of the rat anti-TNF-.alpha. antibody on the first
episode of colitis and on relapse colitis, single-dose
administration of the hamster anti-CD81 antibody exhibited a
stronger inhibitory effect on the first episode of colitis and on
relapse colitis.
(2) Colitis Symptom Score Assessment and Ulcer Score Assessment
[0091] Based on the results of the chronological changes in the
symptoms of colitis in FIG. 1(a) and FIG. 1(b), an average colitis
score of each of the mice from Day 5 to Day 9 was used as the first
episode colitis score, and an average colitis score of each of the
mice from Day 10 to Day 14 was used as the relapse colitis score.
Further, a two group comparison was performed between each group
and the pathological condition control group (hamster IgG
administration group) with respect to the colitis score (first
episode colitis score and relapse colitis score) and the ulcer
score by the Wilcoxon test. The results thus obtained were shown in
Tables 1 and 2.
TABLE-US-00001 TABLE 1 Results of the colitis score First episode
colitis score Relapse colitis score Significant Significant Group
Score (SEM) difference Score (SEM) difference Pathological 0.54
(0.14) -- 0.76 (0.22) -- condition control (Hamster IgG)
Pathological 0.50 (0.15) -- 0.52 (0.17) -- condition control (Rat
IgG) Anti-TNF-.alpha. 0.28 (0.09) No 0.54 (0.25) No antibody
administration Anti-CD81 antibody 0.10 (0.08) * 0.10 (0.08) **
administration Sulfasalazine 0.68 (0.16) No 0.40 (0.13) No
administration Normal 0.00 (0.00) ** 0.00 (0.00) ** * 0.01 < p
< 0.05, ** p < 0.01
TABLE-US-00002 TABLE 2 Results of the ulcer score Ulcer score Group
Score Significant difference Pathological condition control 0.4 --
(Hamster IgG) Pathological condition control 0.3 -- (Rat IgG)
Anti-TNF-.alpha. antibody 0.3 No administration Anti-CD81 antibody
0.0 * administration Sulfasalazine administration 0.1 No Normal 0.0
* * 0.01 < p < 0.05, ** p < 0.01
[0092] As is apparent from the results of Tables 1 and 2, compared
to single-dose administration of the anti-TNF-.alpha. antibody and
daily administration of sulfasalazine, both of which are the
existing therapeutic drugs, single-dose administration of the
anti-CD81 antibody could significantly and potently inhibit the
symptoms of the first episode of colitis and relapse colitis, and
after the first episode of colitis and relapse colitis, potently
inhibit large intestinal ulcer. Accordingly, it was revealed that
single-dose administration of the anti-CD81 antibody exhibited a
long-term remission-maintaining effect on relapse-remitting type
inflammatory bowel disease such as ulcerative colitis.
Example 2
Long-Term Remission Effect of an Anti-CD81 Antibody in the Mouse
Model of Colitis Induced by 2,4,6-trinitrobenzenesulfonic Acid
(TNBS)
[0093] The anti-CD81 antibody was administered once to the mouse
model of relapse-remitting type inflammatory bowel disease (Crohn's
disease and ulcerative colitis), which is obtained by administering
TNBS to a mouse, and the relapse (recurrence)-inhibitory effect of
single-dose administration of the anti-CD81 antibody on
inflammatory bowel disease was examined by comparing with
multiple-dose administration of sulfasalazine, which is the
existing therapeutic drug.
1. Method
(1) Preparation of TNP-OVA
[0094] Into 25 ml of distilled water for injection, 0.5 g of
ovalbumin (OVA: Sigma) and 0.5 g of K.sub.2CO.sub.3 (Nacalai
Tesque, Inc.) were dissolved (OVA solution). Into 25 ml of 0.1 M
K.sub.2CO.sub.3, 0.5 g of 2,4,6-trinitrobenzenesulfonic acid (TNBS:
Nacalai Tesque, Inc.) was dissolved (TNBS solution). The OVA
solution and the TNBS solution were mixed and stirred overnight at
room temperature. Then, the solution after stirring was dialyzed
against 0.01 M NaHCO.sub.3 (Nacalai Tesque, Inc.) through a
dialysis membrane (Spectra/Por Membrane MWCO: 10,000, Spectrum
Medical Industries, Inc.). The solution after dialysis was
subjected to protein quantitation using the BCA protein assay
reagent (Pierce).
(2) Grouping, Sensitization, Challenge, Relapse, and Drug
Administration
[0095] Five-week-old mice (SJL/JorlIcoCrj, male, Charles River
Laboratories Japan, Inc.) were administered (sensitized) with a 1:1
emulsion of complete Freund's adjuvant (CFA: Difco Laboratories)
and 2 mg/ml TNP-OVA by subcutaneous injection in the back at 0.1
ml/head. Seven days after sensitization, a 10 mg/ml solution of
TNBS in 50% ethanol was infused into the intestine under ether
anesthesia (infused at 0.2 ml/head through a sonde that was
inserted up to 3 cm from the anus: challenge). Six days after the
challenge, the mice were divided into four groups, where one group
contained seven mice, according to the body weight and symptom
score. Twenty one days after the grouping, the mice were fasted for
one day, and 23 days after the grouping, a 10 mg/ml solution of
TNBS in 50% ethanol was infused into the intestine again (infused
at 0.2 ml/head through a sonde that was inserted up to 3 cm from
the anus: challenge). The test was completed 28 days after the
grouping.
[0096] Four groups were each assigned as a 1 mg (0.2 ml)/head
anti-CD81 antibody solution (Clone: 2F7, the product of
SouthernBiotech) administration group, a 0.1 mg (0.2 ml)/head
anti-CD81 antibody solution (Clone: 2F7, the product of
SouthernBiotech) administration group, a 0.2 ml/head control
antibody-administered pathological condition control group, and a
200 mg/kg sulfasalazine (SSZ: Sigma) administration group. As the
control antibody, hamster IgG was used.
[0097] As to the drug administration, the respective antibodies
were intraperitoneally administered to the antibody administration
group and to the pathological condition control group once on the
day of grouping, while SSZ was orally administered every day, once
daily, from the day of grouping. The test was completed 28 days
after the grouping. The SSZ solution was suspended in a 0.5%
solution of methyl cellulose (Nacalai Tesque, Inc.).
(3) Symptom Score Assessment
[0098] The symptoms of colitis were observed from Day 15 to Day 28
after the grouping (after single-dose administration of the
anti-CD81 antibody) to examine the persistence of the effect. The
symptoms of intestinal inflammation were assessed by score
according to the condition of stool (normal stool (score: 0), loose
stool (score: 1), and diarrhea (score: 2)). The dead mice were
excluded from the results. For statistical processing, a two group
comparison was performed between the pathological condition control
group and the anti-CD81 antibody administration group or the SSZ
administration group by the Wilcoxon rank sum test.
2. Results
[0099] An average colitis symptom score of each group from Day 15
to Day 28 after the grouping (after single-dose administration of
the anti-CD81 antibody) and the standard error (SEM) were shown in
Table 3. Also, in FIG. 2, the chronological changes in the symptoms
of colitis in the hamster IgG single-dose administration group, the
1 mg (0.2 ml)/head anti-CD81 antibody single-dose administration
group, and the sulfasalazine daily administration group were
shown.
TABLE-US-00003 TABLE 3 Results of the colitis score assessment
Symptoms of colitis Group (SEM) Pathological condition control 1.16
(0.34) Anti-CD81 antibody administration 0.61 (0.16)* (Single-dose
administration; 1 mg/head) Anti-CD81 antibody administration 0.84
(0.12) (Single-dose administration; 0.1 mg/head) SSZ administration
0.87 (0.09) (Daily multiple-dose administration; 200 mg/kg) *0.01
< p < 0.05, **p < 0.01
[0100] From the results of Table 3, it was revealed that the
ameliorating effect on the symptoms of intestinal inflammation in
the TNBS-induced colitis mice persisted from Day 15 to Day 28 after
single-dose administration of the anti-CD81 antibody (1 mg/head).
Also, from the results of FIG. 2, it was revealed that the
anti-CD81 antibody exerted a long-term remission-maintaining effect
in the inflammatory bowel disease model by single-dose
administration.
Example 3
Relapse-Inhibitory Effect of Single-Dose Administration of an
Anti-CD81 Antibody in the Mouse Model of Colitis Induced by an
Aqueous Solution of Dextran Sulfate (DSS)
[0101] The anti-CD81 antibody was administered once to the mouse
model of relapse-remitting type ulcerative colitis, which is
obtained by administering an aqueous solution of dextran sulfate
(DSS) to a mouse, and the relapse-inhibitory effect of the
anti-CD81 antibody on relapse-remitting type ulcerative colitis
that was induced for a longer period of time was examined.
1. Method
(1) Preparation of DSS
[0102] Dextran sulfate (the product of Wako Pure Chemical
Industries, Ltd., an average molecular weight of 5,000) was
dissolved in drinking water to prepare a 2% aqueous solution.
(2) Preparation of an Administration Solution
[0103] As the test drug to be studied, a hamster anti-CD81 antibody
was used, and as the pathological condition control test drug,
hamster IgG was used. The concentrated original solution of the
hamster anti-CD81 antibody (Clone: 2F7, the product of
SouthernBiotech) was used as the administration solution, while
phosphate buffer was added to the original solution of hamster IgG
to prepare a 0.25 mg/ml administration solution.
(3) Grouping, DSS Ingestion, and Drug Administration
[0104] Four-week-old mice (BALB/cAnNCrlCrlj, male, the product of
Charles River Laboratories Japan, Inc.) were purchased, and the
mice were reared for quarantine for seven days, and then reared
conventionally for seven days. Thereafter, the mice were measured
for body weight and divided into two groups, where one group
contained six mice. On the day of grouping, hamster IgG or the
anti-CD81 antibody was intraperitoneally administered once at 0.5
mg/mouse. All mice were fed 2% DSS ad libitum (first DSS ingestion)
for five days after the grouping, and then normal drinking water
for the next 10 days. Subsequently, all mice were fed 2% DSS ad
libitum (second DSS ingestion) for five days, and then normal
drinking water for the next nine days. Subsequently, all mice were
fed 2% DSS ad libitum (third DSS ingestion) for five days, and then
normal drinking water for the next 16 days. Subsequently, all mice
were fed 2% DSS ad libitum (forth DSS ingestion) for seven days,
and the test was completed.
(4) Colitis Symptom Score Assessment
[0105] From the day of grouping to the day of completion of the
test, the symptoms of colitis were observed. The symptoms of
colitis were assessed by score according to the condition of stool
(normal stool (score: 0), loose stool (score: 1), and diarrhea
(score: 2)).
2. Results
[0106] The chronological changes in the symptoms of colitis by
colitis symptom score assessment in the mice that received
single-dose administration of either one of hamster IgG or the
hamster anti-CD81 antibody were shown in FIG. 3. In FIG. 3, Day 0
corresponds to the day of grouping, Day 0 to Day 5 correspond to
the first DSS ingestion period, Day 15 to Day 20 correspond to the
second DSS ingestion period, Day 29 to Day 34 correspond to the
third DSS ingestion period, and Day 50 to Day 56 correspond to the
fourth DSS ingestion period. As is apparent from FIG. 3, the first
episode of DSS-induced colitis was inhibited, and moreover, the
subsequent three recurrences of DSS-induced colitis were inhibited
and ameliorated by only single-dose administration of the anti-CD81
antibody on Day 0, whereby it exhibited a long-term
remission-maintaining effect.
Example 4
Relapse-Inhibitor Effect of Single-Dose Administration of an
Anti-CD81 Antibody in the Mouse Model of Colitis Induced by an
Aqueous Solution of Dextran Sulfate (DSS)
[0107] The anti-CD81 antibody clone 2F7 or clone Eat2 was
administered once to the mouse model of relapse-remitting type
ulcerative colitis, which is obtained by administering an aqueous
solution of dextran sulfate (DSS) to a mouse, and the
relapse-inhibitory effect of each clone of the anti-CD81 antibody
on relapse-remitting type ulcerative colitis that was induced
multiple times was examined.
1. Method
(1) Preparation of DSS
[0108] Dextran sulfate (the product of Wako Pure Chemical
Industries, Ltd., an average molecular weight of 5,000) was
dissolved in drinking water to prepare a 2% aqueous solution.
(2) Preparation of an Administration Solution
[0109] As the test drug to be studied, two kinds of hamster
anti-CD81 antibodies were used, and as the pathological condition
control test drug, hamster IgG was used.
[0110] The concentrated original solutions of the hamster anti-CD81
antibodies (Clone: 2F7, the product of SouthernBiotech or Clone:
Eat2, the product of BioLegend, Inc., both of which were the
anti-mouse CD81 monoclonal antibodies) were used as the
administration solution, while phosphate buffer was added to the
original solution of hamster IgG to prepare a 0.1 mg/ml
solution.
(3) Grouping, DSS Ingestion, and Drug Administration
[0111] Four-week-old mice (BALB/cAnNCrlCrlj, male, the product of
Charles River Laboratories Japan, Inc.) were purchased. The mice
were reared for quarantine for seven days and then measured for
body weight, and divided into four groups, where one group
contained 15 mice. On the day of grouping, hamster IgG the
anti-CD81 antibody clone 2F7 or the anti-CD81 antibody clone Eat2
were intraperitoneally administered once at 0.2 mg/mouse. The mice
were fed 2% DSS ad libitum (first DSS ingestion) for five days
after the grouping, and then normal drinking water for the next 10
days. Subsequently, the mice were fed 2% DSS ad libitum (second DSS
ingestion) for five days, and then normal drinking water for the
next 10 days. Subsequently, the mice were fed 2% DSS ad libitum
(third DSS ingestion) for five days, and then normal drinking water
for the next six days. The normal group was fed normal drinking
water throughout the experimental period.
(4) Colitis Symptom Score Assessment
[0112] The symptoms of colitis were observed for 40 days from the
day of grouping. The symptoms of colitis were assessed by score
according to the condition of stool (normal stool (score: 0), loose
stool (score: 1), and diarrhea (score: 2)).
2. Results
[0113] The chronological changes in the symptoms of colitis by
colitis symptom score assessment in the mice that received
single-dose administration of hamster IgG or either one of the
hamster anti-CD81 antibody clone 2F7 and the anti-CD81 antibody
clone Eat2 were shown in FIG. 4. From FIG. 4, it was observed that
the first episode of DSS-induced colitis was inhibited, and
further, the subsequent two recurrences of DSS-induced colitis were
inhibited by only single-dose administration of the anti-CD81
antibody clone 2F7 or the anti-CD81 antibody clone Eat2 on Day 0,
whereby it exhibited a long-term remission-maintaining effect.
[0114] Based on the results of the chronological changes in the
symptoms of colitis in FIG. 4, an average colitis score of each of
the mice from Day 0 to Day 11 was used as the first episode colitis
score, an average colitis score of each of the mice from Day 15 to
Day 23 was used as the relapse colitis score, and an average
colitis score of each of the mice from Day 30 to Day 38 was used as
the second relapse colitis score. Then, a two group comparison was
performed between each group and the pathological condition control
group (hamster IgG administration group) with respect to the
colitis score (first episode colitis score, relapse colitis score,
and second relapse colitis score) by the Wilcoxon test. The results
thus obtained were shown in Table 4.
TABLE-US-00004 TABLE 4 Results of the colitis score assessment
Symptoms of colitis(SEM) First episode Relapse Second relapse
colitis score colitis score colitis score Group (Day 0-11) (Day
15-23) (Day 30-38) Pathological condition 0.38 (0.08) 0.64 (0.08)
0.55 (0.09) control Single-dose 0.11 (0.03)* 0.31 (0.09)* 0.21
(0.06)* administration of the anti-CD81 antibody 2F7 (0.2 mg/head)
Single-dose 0.04 (0.02)** 0.23 (0.05)** 0.21 (0.07)* administration
of the anti-CD81 antibody Eat2 (0.2 mg/head) Normal mouse 0.00
(0.00)** 0.00 (0.00)** 0.00 (0.00)** *0.01 < p < 0.05, **p
< 0.01
[0115] From the results of Table 4, it was revealed that the
persistent ameliorating effect on the symptoms of intestinal
inflammation was obtained after single-dose administration of the
anti-CD81 antibody (0.2 mg/head) as evidenced by the first episode
colitis score, relapse colitis score, and second relapse colitis
score.
INDUSTRIAL APPLICABILITY
[0116] The present invention revealed that refractory inflammatory
bowel disease could be prevented, ameliorated, and treated by
administration of an anti-CD81 antibody, and an anti-CD81 antibody
had a long-term remission-maintaining effect even by single-dose
administration. In other words, the present invention revealed that
an anti-CD81 antibody exerted a profound effect even in a patient
with refractory inflammatory bowel disease (IBD) in whom
single-dose administration or even multiple-dose administration of
an existing therapeutic agent for inflammatory bowel disease has
achieved only as far as a remission-inducing effect.
[0117] Accordingly, a drug containing an anti-CD81 antibody as the
active ingredient exhibits not only a remission-inducing effect on
inflammatory bowel disease, but also a long-term
remission-maintaining effect even by single-dose administration,
and thus is utilizable as a drug for maintaining remission of
inflammatory bowel disease and as a preventive, ameliorating, or
therapeutic drug for inflammatory bowel disease.
Sequence CWU 1
1
211497DNAHomo sapiens 1gagcgagcgc gcaacggcgg cgacggcggc gaccccaccg
cgcatcctgc caggcctccg 60gcgcccagcg ccccacgcgc ccccgcgccc ccgcgccccc
gcgccccttt cttcgcgccc 120ccgcccctcg gcccgccagg cccccttgcc
ggccacccgc caggccccgc gccggcccgc 180ccgccgccca ggaccggccc
gcgccccgca ggccgcccgc cgcccgcgcc gccatgggag 240tggagggctg
caccaagtgc atcaagtacc tgctcttcgt cttcaatttc gtcttctggc
300tggctggagg cgtgatcctg ggtgtggccc tgtggctccg ccatgacccg
cagaccacca 360acctcctgta tctggagctg ggagacaagc ccgcgcccaa
caccttctat gtaggcatct 420acatcctcat cgctgtgggc gctgtcatga
tgttcgttgg cttcctgggc tgctacgggg 480ccatccagga atcccagtgc
ctgctgggga cgttcttcac ctgcctggtc atcctgtttg 540cctgtgaggt
ggccgccggc atctggggct ttgtcaacaa ggaccagatc gccaaggatg
600tgaagcagtt ctatgaccag gccctacagc aggccgtggt ggatgatgac
gccaacaacg 660ccaaggctgt ggtgaagacc ttccacgaga cgcttgactg
ctgtggctcc agcacactga 720ctgctttgac cacctcagtg ctcaagaaca
atttgtgtcc ctcgggcagc aacatcatca 780gcaacctctt caaggaggac
tgccaccaga agatcgatga cctcttctcc gggaagctgt 840acctcatcgg
cattgctgcc atcgtggtcg ctgtgatcat gatcttcgag atgatcctga
900gcatggtgct gtgctgtggc atccggaaca gctccgtgta ctgaggcccc
gcagctctgg 960ccacagggac ctctgcagtg ccccctaagt gacccggaca
cttccgaggg ggccatcacc 1020gcctgtgtat ataacgtttc cggtattact
ctgctacacg tagccttttt acttttgggg 1080ttttgttttt gttctgaact
ttcctgttac cttttcaggg ctgacgtcac atgtaggtgg 1140cgtgtatgag
tggagacggg cctgggtctt ggggactgga gggcaggggt ccttctgccc
1200tggggtccca gggtgctctg cctgctcagc caggcctctc ctgggagcca
ctcgcccaga 1260gactcagctt ggccaacttg gggggctgtg tccacccagc
ccgcccgtcc tgtgggctgc 1320acagctcacc ttgttccctc ctgccccggt
tcgagagccg agtctgtggg cactctctgc 1380cttcatgcac ctgtcctttc
taacacgtcg ccttcaactg taatcacaac atcctgactc 1440cgtcatttaa
taaagaagga acatcaggca tgctaccagg cctgtgcagt ccctcag 14972236PRTHomo
sapiens 2Met Gly Val Glu Gly Cys Thr Lys Cys Ile Lys Tyr Leu Leu
Phe Val 1 5 10 15 Phe Asn Phe Val Phe Trp Leu Ala Gly Gly Val Ile
Leu Gly Val Ala 20 25 30 Leu Trp Leu Arg His Asp Pro Gln Thr Thr
Asn Leu Leu Tyr Leu Glu 35 40 45 Leu Gly Asp Lys Pro Ala Pro Asn
Thr Phe Tyr Val Gly Ile Tyr Ile 50 55 60 Leu Ile Ala Val Gly Ala
Val Met Met Phe Val Gly Phe Leu Gly Cys 65 70 75 80 Tyr Gly Ala Ile
Gln Glu Ser Gln Cys Leu Leu Gly Thr Phe Phe Thr 85 90 95 Cys Leu
Val Ile Leu Phe Ala Cys Glu Val Ala Ala Gly Ile Trp Gly 100 105 110
Phe Val Asn Lys Asp Gln Ile Ala Lys Asp Val Lys Gln Phe Tyr Asp 115
120 125 Gln Ala Leu Gln Gln Ala Val Val Asp Asp Asp Ala Asn Asn Ala
Lys 130 135 140 Ala Val Val Lys Thr Phe His Glu Thr Leu Asp Cys Cys
Gly Ser Ser 145 150 155 160 Thr Leu Thr Ala Leu Thr Thr Ser Val Leu
Lys Asn Asn Leu Cys Pro 165 170 175 Ser Gly Ser Asn Ile Ile Ser Asn
Leu Phe Lys Glu Asp Cys His Gln 180 185 190 Lys Ile Asp Asp Leu Phe
Ser Gly Lys Leu Tyr Leu Ile Gly Ile Ala 195 200 205 Ala Ile Val Val
Ala Val Ile Met Ile Phe Glu Met Ile Leu Ser Met 210 215 220 Val Leu
Cys Cys Gly Ile Arg Asn Ser Ser Val Tyr 225 230 235
* * * * *
References